cure of mammary carcinomas in her-2 transgenic mice...

Cure of Mammary Carcinomas in Her-2 Transgenic Mice through

Sequential Stimulation of Innate (Neoadjuvant Interleukin-12)

and Adaptive (DNA Vaccine Electroporation) Immunity

Michela Spadaro,1Elena Ambrosino,

1

Manuela Iezzi,2Emma Di Carlo,

2

Pamela Sacchetti,1Claudia Curcio,

1

Augusto Amici,3Wei-Zen Wei,

4Piero Musiani,

2

Pier-Luigi Lollini,5Federica Cavallo,

1and

Guido Forni1

1Department of Clinical and Biological Sciences, University of Turin,Orbassano, Italy; 2Center of Excellence on Aging Research,‘‘G. D’Annunzio’’ University Foundation, Chieti, Italy; 3Department ofMolecular, Cellular and Animal Biology, University of Camerino,Camerino, Italy; 4Karmanos Cancer Institute, Wayne State University,Detroit, Michigan; and 5Cancer Research Section, Department ofExperimental Pathology, University of Bologna, Bologna, Italy

ABSTRACT

Purpose: Whereas neoadjuvant therapy is emerging as

a treatment option in early primary breast cancer, no data

are available on the use of antiangiogenic and immuno-

modulatory agents in a neoadjuvant setting. In a model of

Her-2 spontaneous mammary cancer, we investigated the

efficacy of neoadjuvant interleukin 12 (IL-12) followed by

‘‘immune-surgery’’ of the residual tumor.

Experimental Design: Female BALB/c mice transgenic

for the rat Her-2 oncogene inexorably develop invasive

carcinomas in all their mammary glands by the 23rd week of

age. Mice with multifocal in situ carcinomas received four

weekly i.p. injections of 100 ng IL-12 followed by a 3-week

rest. This course was given four times. A few mice

additionally received DNA plasmids encoding portions of

the Her-2 receptor electroporated through transcutaneous

electric pulses.

Results: The protection elicited by IL-12 in combina-

tion with two DNA vaccine electroporations kept 63% of

mice tumor-free. Complete protection of all 1-year-old mice

was achieved when IL-12-treated mice received four

vaccine electroporations. Pathologic findings, in vitro tests,

and the results from immunization of both IFN-; and

immunoglobulin gene knockout transgenic mice and of

adoptive transfer experiments all show that IL-12 augments

the B- and T-cell response elicited by vaccination and

slightly decreases the number of regulatory T cells. In

addition, IL-12 strongly inhibits tumor angiogenesis.

Conclusions: In Her-2 transgenic mice, IL-12 impairs

tumor progression and triggers innate immunity so markedly

that DNA vaccination becomes effective at late points in time

when it is ineffective on its own.

INTRODUCTION

As breast cancer is the most common primary malignant

disease in women and often associated with poor prognosis, new

strategies for its cure and prevention are urgently needed. There

has been a surge of interest in neoadjuvant medical therapy for

early breast cancer over the last 10 years in the light of the

experience in locally advanced inoperable breast cancer, where

systemic treatment before local therapy is now the standard of

care (1). Randomized trials comparing neoadjuvant medical and

conventional postoperative adjuvant therapy have shown similar

rates of local control and overall survival (1–3). These results

have been obtained with both chemotherapy and endocrine

neoadjuvant therapy, whereas no data are available on the use of

antiangiogenic and immunomodulatory agents in a neoadjuvant

setting. Microvessel density is an independent and highly

significant prognostic factor in breast cancer. Antiangiogenic

management designed to prevent the sprouting of new vessels

restrains tumor expansion (4). However, although antiangiogenic

therapy significantly delays early tumor progression, its

contribution to tumor cure is not apparent (5, 6).

Interleukin 12 (IL-12) is a cytokine with a potent

antiangiogenic and immunomodulatory activity that elicits

numerous downstream proinflammatory cytokines and danger

signals, and leads to activation of professional antigen-

presenting cells (7). IFN-g and other downstream cytokines

thus elicited inhibit tumor cells, activate potent antiangiogenic

mechanisms, and even switch the genetic program of a tumor

from proangiogenic to antiangiogenic (7, 8). In many

transplantable tumor models, the sum of these activities results

in significant inhibition (9, 10). In mice transgenic for the

activated rat (r) Her-2 oncogene (neu and ErbB-2 in humans)

under the control of the mouse mammary tumor virus promoter

(BALB-neuT), IL-12 delays early carcinogenesis progression,

but is not enough to cure established tumors (11, 12). However,

it also acts as an essential component of a combined cell

vaccine that prevents the early onset of carcinomas (13).

In these mice, adaptive immunity elicited by vaccination

with DNA plasmids coding the transmembrane and extracellular

domains of r-Her-2 (TMEC plasmids) clears early in situ

carcinomas and keeps most mice tumor-free (14). This

prevention is enhanced when DNA vaccination is boosted by

Received 9/13/04; revised 11/19/04; accepted 12/6/04.Grant support: Italian Association for Cancer Research, ItalianMinistries for the Universities and Health, University of Turin,Compagnia di San Paolo, Turin, and Center of Excellence on Aging,University of Chieti, Italy.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Federica Cavallo, Department of Clinical andBiological Sciences, University of Turin, Ospedale San Luigi Gonzaga,Regione Gonzole 10, I-10043 Orbassano, Italy. Phone: 39-11-790-5419;Fax: 39-11-236-5417; E-mail: [email protected].

D2005 American Association for Cancer Research.

Vol. 11, 1941–1952, March 1, 2005 Clinical Cancer Research 1941

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r-Her-2+ IFN-g-releasing allogeneic cells (15), or when IL-1hfragment (16) or soluble LAG-3 costimulatory molecule (17) are

administered concurrently with the vaccine. Moreover, complete

prevention is afforded by repeated plasmid electroporations (18).

All these cell and DNA vaccines, however, are ineffective

when an invasive carcinoma is established. In the study

described in this paper, we determined whether early neo-

adjuvant stimulation of innate immunity by IL-12 could render

effective subsequent DNA vaccination after the onset of

carcinomas. The results show that early IL-12 treatment of

BALB-neuT mice makes DNA vaccination effective, whereas it

is ineffective on its own. This suggests that sequential

combination of neoadjuvant stimulation of innate immunity

followed by ‘‘immune surgery’’ of the residual tumor cells

through induction of adaptive immunity can be proposed as a

new and effective strategy.

MATERIALS AND METHODS

Mice. BALB-neuT mice were bred under specific

pathogen-free conditions by Charles River (Calco, Italy; ref. 5).

BALB-neuT mice knocked out for the IFN-g (BALB-neuT/

IFNgKO), and BALB-neuT mice knocked out for the

immunoglobulin (Ig) l chain gene (BALB-neuT/AKO) were

generated by crossing BALB-neuT mice with BALB/c mice KO

for the IFNc gene from The Jackson Laboratory (Bar Harbor,

ME), or BALB/c KO mice for the Ig A chain kindly provided by

Dr. T. Blankenstein (Free University, Berlin, Germany; ref. 19).

Mice were randomly assigned to control and treatment groups

and all groups were treated concurrently. As each experiment

was repeated twice to thrice with similar results, data were

cumulated and reported. Mammary glands were inspected at

weekly intervals to note tumor appearance and tumor masses

were measured with calipers in two perpendicular diameters.

Progressively growing masses >1 mm mean diameter were

regarded as tumors. Tumor multiplicity was calculated as the

cumulative number of incident tumors/total number of mice and

is reported as mean F SE (18). Mice were treated according to

the European Union guidelines.

Interleukin-12 Administration. Recombinant mouse

IL-12 (kindly provided by Dr. Stan Wolf, Genetics Institute,

Cambridge, MA) was diluted in PBS supplemented with 0.01%

mouse serum albumin (MSA; Sigma, St. Louis, MO) and

administered i.p. as previously described (12). Starting from the

7th (7wMSA or 7wIL12) or the 14th (14wIL12) week of age,

BALB/c and BALB-neuT mice received a course of one

weekly i.p. injection of 0.2 mL PBS containing MSA only

(MSA controls) or MSA plus 100 ng of IL-12, for 4 weeks,

followed by a 3-week rest. This course was given four times in

7wMSA and 7wIL12 or thrice only in 14wIL12. Other groups

of mice were not treated. Because no appreciable differences in

tumor growth rate and pathologic findings were found between

the untreated mice and the MSA controls, only the data of one

of these two groups are shown in Results.

Whole-Mount Image Analyses. Whole mounts of all

mammary glands were done as indicated in http://ccm.ucdavis.edu/

tgmouse/HistoLab/wholmt1.htm. Digital images were acquired

by dividing the whole mount of each gland into 10 quadrants.

Ten points were randomly chosen on the duct surface in each

quadrant and the corresponding lesions were measured. All

lesions with a diameter >150 Am on the same quadrant were

counted. Images of whole-mount preparations were taken with a

Nikon Coolpix 950 digital camera (Nital Spa, Turin, Italy)

mounted on a stereoscopic Leica MZ 6 microscope (Leica

Microsystems, Milan, Italy). A 0.63 objective were used to

obtain images with a total magnification of �630 and a

resolution of 1,600 � 1,200 pixels. Images were acquired within

Adobe Photoshop version 6.0 graphic software (Adobe Systems,

San Jose, CA). Glands larger than a single imaging area were

captured by photographing contiguous microscopic fields in a

raster pattern. Each captured image was merged using the layer

technique in Adobe Photoshop to form a single composite image

for analysis. Spatial calibration was determined by photographing

a 1 mm stage using the same parameters as those for image

capturing of whole-mount preparations. The distance drawn on

the 1 mm calibration image was divided by 1,000 to find the

number of pixels per micrometer. In each image, 100 discrete

points were randomly chosen on the duct surface and lesion

widths in micrometer were measured perpendicular to duct

direction. Points with no lesion were ranked as zero. Lesion

measurements were recorded on an Excel spreadsheet and mean

and SE were calculated for each treatment group. Statistics were

obtained with a two-tailed Student’s t test.

Preparation of DNA Plasmids. TMEC plasmids were

produced as previously described (14–18). For DNA electro-

poration, 25 Ag of TMEC plasmids in 20 AL 0.9% of NaCl

with 6 mg/mL polyglutamate were injected bilaterally into the

tibial muscle of the hind legs of anesthetized mice. Transcu-

taneous electric pulses were applied on the shaved skin.

Square-wave electric pulses were generated by a T820 electro-

porator (BTX, San Diego, CA). Two 25 ms pulses with a field

strength of 375 V/cm were administered (18).

Evaluation of Serum Vascular Endothelial Growth

Factor. Blood samples from groups of three control or treated

BALB-neuT mice were collected and the presence of vascular

endothelial growth factor in each serum was evaluated with a

sandwich ELISA (R&D Systems Inc., Minneapolis, MN).

India Ink Blood Vessel Perfusion and Image Analysis.

Groups of three mice were anesthetized and perfused via the left

ventricle. An initial blood washout with a solution of 0.5%

sodium nitrite and 10 units/mL heparin in PBS at 37jC was

immediately followed by reperfusion with black India ink. Fat

pads were prepared as whole mounts and stained with carmine.

Images of whole-mount preparations were taken as described

above with a 4.3 objective giving a total magnification of �430.

At least 10 images were taken for each mammary gland and 25

images from each mouse were randomly chosen for image

analysis. The area of the black blood vessel was selected with the

Magic Wand Tool in Adobe Photoshop and the number of pixels

recorded on an Excel spreadsheet. The width of the blood vessel

network is calculated as a percent of total image area.

Histology and Immunohistochemistry. Groups of three

mice were sacrificed at the indicated times and samples of

mammary tissue or spleen were processed as previously described

for immunohistochemical and histologic analysis (20). For

immunohistochemistry, acetone-fixed cryostat sections were

incubated for 30 minutes with antibodies anti-Mac-1 (CD11b/

CD18, clone M1/70.5), anti-CD8 (Ly/T2, clone YT5 169.4) and

Neoadjuvant IL-12 and DNAVaccine Electroporation1942



anti-CD4 (LT34, clone YT5.191.1.2; all from Sera-Lab, Crawley

Down, Sussex, United Kingdom), or anti-CD11c (clone N418;

Chemicon International Inc., Temecula, CA), anti-CD40L

(CD154, gp39; clone H-215; Santa Cruz, Biotechnology Inc.,

Santa Cruz, CA), anti-CD86 (B72, clone PO3), and anti-CD45R/

B220 (clone RA3-6B2; all from PharMingen, San Diego, CA),

anti-IFN-g (clone XMG1.2, kindly provided by Dr. S. Landolfo,

University of Turin, Turin, Italy). To evaluate the expression of

r-p185neu and proliferating cell nuclear antigen, paraffin-embed-

ded sections were tested with polyclonal rabbit anti-Her-2

antiserum (C-18, Santa Cruz Biotechnology) and anti–prolifer-

ating cell nuclear antigen (Ylem, Rome, Italy) antibody. After

washing, sections were overlaid with biotinylated goat anti-rat,

anti-hamster, and anti-rabbit or horse anti-goat Ig (Vector

Laboratories, Burlingame, CA) for 30 minutes and incubated

with avidin-biotin complex method complex/alkaline phospha-

tase (DAKO, Glostrup, Denmark). To quantify germinal centers

(GC), each spleen was transversely dissected into four segments

from which semiserial sections were obtained. The GC in the total

area of each section were counted. Morphologic studies were

conducted independently by three pathologists in a blind fashion.

Antibody Response. Sera obtained at progressive time

points from mice of each treatment group were diluted 1:100 in

PBS-azide-bovine serum albumin (Sigma) and the presence of

anti-r-p185neu antibody was determined by flow cytometry using

BALB/c 3T3 fibroblasts, wild-type, or stably cotransfected with

the wild-type r-Her-2, mouse class I H-2Kd and B7.1 genes

(BALB/c 3T3-NKB). FITC-conjugated goat anti-mouse anti-

body specific for mouse IgG Fc (DAKO) was used to detect

bound primary antibody. Normal mouse serum was the negative

control. The monoclonal antibody Ab4 (Oncogene Research

Products, Cambridge, MA), which recognizes an extracellular

domain of r-p185neu, was used as a positive control. Serial Ab4

dilutions were used to generate a standard curve to determine the

concentration (Ag/mL) of anti-r-p185neu antibody in serum (12).

For isotype determinations, sera from 20-week-old mice were

diluted 1:20 and incubated with BALB/c 3T3 and BALB/c 3T3-

NKB. Cells were then incubated for 30 minutes with rat biotin-

conjugated antibody anti-mouse IgA, IgM, IgG1, IgG2a, IgG2b,

IgG3 (Caltag Laboratories, Burlingame, CA). After washing,

cells were incubated with 5 AL streptavidin-phycoerythrin (PE;

DAKO), and resuspended in PBS-azide-bovine serum albumin

containing 1 mg/mL propidium iodide to gate dead cells. Flow

cytometry was done with a FACScan (Becton Dickinson,

Mountain View, CA). Data were analyzed through CELLQuest

(Becton Dickinson) software.

Antibody-Dependent Cellular Cytotoxicity. TUBO

cells are a cloned cell line from a carcinoma that arose in a

Fig. 1 Whole mounts of the mamma-ry gland documenting the modulationof carcinogenesis in 7wIL12-treatedBALB-neuT mice. Numerous neoplas-tic side bud lesions (dark spots pointedby the arrows) are scattered all over theglands of 7-week-old BALB-neuTmice (A ). Mice with these lesionsreceived an i.p. injection of MSA onlyor MSA + 100 ng of IL-12 weekly for 4weeks, followed by 3 weeks of rest.These courses were repeated four times(7wMSA; 7wIL12; B). In 7wMSA-treated control mice, the side buds aremore diffuse and larger at week 16 (C)and coalesce to formmultiple masses atweek 20 (E). In 7wIL12-treatedmice, amarked reduction in both the size anddiffusion of these side buds is evident atweek 16 and week 20 (D). At week 20,confinement of the neoplastic lesionsclose to the nipple is a typical finding.However, the lesions are much largerthan at week 16 (F). LN, mammarylymph node. (A -E , magnification�63). Three different mice were usedfor each treatment.

Clinical Cancer Research 1943



BALB-neuT mouse (14). Five thousand [3H]dTh-labeled TUBO

cells were incubated for 2 hours at 4jC with progressive

dilutions (1:10 to 1:100) of sera in HBSS (Sigma) and then

washed. Effector spleen cells (Spc) from untreated mice were

then added at 50:1 effector-to-target ratio to each well of round-

bottom microtiter plates (Nunc, Roskilde, Denmark) in triplicate

and lysis was determined as previously described (21).

IFN-; Production. Spc (1 � 107) were stimulated with

5 � 105 mitomycin-C (Sigma) treated TUBO cells in the

presence of 10 units/mL recombinant IL-2 (Eurocetus, Milan,

Italy). After 4 days, Spc were recovered, washed, and seeded at

2 � 106 cells/mL in the presence of anti-CD28 and anti-CD3

(1 Ag/mL final concentration, PharMingen) for 3 hours. IFN-g-

producing cells were identified by using the mouse IFN-g cell

enrichment and detection kit (Miltenyi Biotec, Bergisch-

Gladbach, Germany). Recovered Spc were labeled with an

anti-IFN-g (clone R4-6A2) conjugated with an anti-CD45 (clone

30S11) monoclonal antibody (Miltenyi Biotec) for 5 minutes on

ice, then incubated for 45 minutes at 37jC. Cross-staining was

avoided by keeping the cell density at 1 � 105 cells/mL. IFN-g

bound to the capture matrix was stained with PE-conjugated

monoclonal antibody against IFN-g (clone AN.18.17.24,

Miltenyi Biotec). PE-IFN-g-stained cells enriched with anti-PE

microbeads were separated with an autoMACS separator

(Miltenyi Biotec). The cells were counterstained with monoclo-

nal antibody against CD4 or CD8a-FITC (clone YTS 191.1.2,

clone YTS 169 AG 101 HL respectively; Cedarlane, Hornby,

Ontario, Canada) and analyzed by flow cytometry.

Cytotoxicity Assays. Spc (1 � 107) were stimulated for

6 days with 5 � 105 mitomycin-C (Sigma) treated TUBO

cells in the presence of 10 units/mL recombinant IL-2

(Eurocetus) and assayed in a 48-hour [3H]dTh release assay

at effector-to-target ratio from 50:1 to 6:1 in round-bottomed,

96-well microtiter plates in triplicate (21). The results are

expressed as lytic units (LU)20/107 effector cells, with LU20

defined as the number of effector cells needed to kill 20% of

the target cells (22).

Adoptive Transfer. Sera and Spc were collected from

20-week-old 7wIL12 + w16-18TMEC BALB-neuT mice. Spc

were labeled with a PE-conjugated anti-CD90 monoclonal

antibody (clone 53-2.1, PharMingen) and then anti-PE micro-

beads (Miltenyi Biotec) were used to isolate CD90+ cells with an

autoMACS separator (Miltenyi Biotec). Pooled sera (0.5 mL) or

Fig. 2 IL-12 in combination with TMEC plasmid electroporationinhibits the progression of carcinogenesis. Percentage of tumor-free mice(A) and tumor multiplicity (B) in the w16-18pcDNA3 (o, 18 mice),w16-18TMEC (., 9 mice), 7wIL12 (n, 7 mice), and 7wIL12 + w16-18TMEC (5, 8 mice) groups. Arrows, week in which IL-12 was injected;arrowheads, week in which plasmids were electroporated. The tumor-freesurvival curve of 7wIL12 + w16-18TMEC mice is significantly differentfrom that of w16-18pcDNA3 and w16-18TMEC mice (A ; Mantel-Haenszel test, P < 0.0001). The mean tumor multiplicity is significantlylower in 7wIL12 + w16-18TMEC than in w16-18TMEC and w16-18pcDNA3 mice (B ; Student’s t test, P < 0.0006). At the 20th week ofage, the whole mounts of the mammary glands of w16-18pcDNA3 andw16-18TMEC mice show a few small residual side buds (arrows),mostly confined to the nipple region (arrowhead). At week 34, theselesions are larger (arrows), suggesting recommencement of tumorgrowth (D). Oval central black area (LN), lymph node (C and D ,magnification �63). These experiments were done twice.




1 � 107, 90% to 92% CD90+ Spc were administered i.v. in

0.2 mL HBSS to recipient mice at the 10th and 12th week of age.

Recipient mice were sacrificed at the 16th week of age and the

whole mounts of all 10 mammary glands were evaluated by

computer-aided image analysis.

CD4+/CD25+ T Cells. CD4+ Spc obtained at 20 weeks

of age from six mice from each treatment group were purified

with the CD4+ T-cell isolation kit (Miltenyi Biotec), separated

with an autoMACS separator (Miltenyi Biotec), and then

stained with anti-CD4-PE (Cedarlane) and anti-CD25-FITC

antibody (clone 7D4, PharMingen). The cells were resuspended

in PBS-azide-bovine serum albumin containing 1 mg/mL of

propidium iodide to gate dead cells. Flow cytometry was done

with a FACScan (Becton Dickinson). Data were analyzed

through CELLQuest (Becton Dickinson) software, and reported

as the mean of the percentage of CD4+/CD25+ cells determined

in three independent experiments.

Statistics. Differences in tumor incidence were evaluated

with the Mantel-Haenszel log-rank test, and those in tumor

multiplicity, LU20, IFN-g-producing Spc, and antibody titer with

the two-tailed Student’s t test.

RESULTS

IL-12 Slows Down Mammary Carcinogenesis. Mam-

mary carcinogenesis follows a very consistent course in virgin

female BALB-neuT mice (Fig. 1A; ref. 11). When mice with

lesions equivalent to atypical hyperplasia and in situ carcinoma

received recombinant mouse IL-12 i.p. starting from the 7th

week of age (7wIL12; Fig. 1B), a marked reduction in the

progression was already evident at week 16 (Fig. 1C versus D)

and became remarkable at week 20 (Fig. 1E versus F) when

neoplastic side buds were typically confined to the tissue close to

the nipple. However, at week 20, the presence of markedly larger

lesions (Fig. 1D versus F) indicates that the protection has

vanished and that progression has recommenced. No inhibition

was found when IL-12 treatment was postponed to week 14

(14wIL12, not shown).

DNA Vaccination by Electroporation Combined with

IL-12 Inhibits Mammary Carcinogenesis. Electroporation

in vivo of TMEC plasmids when mice display multifocal in situ

carcinomas inhibits carcinogenesis progression (18), but not if

it is postponed to weeks 16 and 18 (w16-18TMEC) when mice

already display invasive carcinomas (Fig. 2A). By contrast, the

combination of 7wIL12 with w16-18TMEC kept 63% of mice

free from palpable tumors until 35 weeks when the experiments

were ended. Extension of the tumor-free survival was

accompanied by a strong reduction in tumor multiplicity (Fig.

2B), whereas the whole mounts displayed only a few small

residual side buds near the nipple at 20 weeks (Fig. 2C). These

lesions are markedly smaller than those of mice that received

7wIL12 only (Fig. 2C versus Fig. 1F). However, at week 35,

the presence of larger side buds again points to slow

recommencement of progression (Fig. 2D). By contrast, all

mice remained free of palpable tumors (Fig. 3A and B) and

their whole mounts displayed no signs of tumor progression

(not shown) when 7wIL12 + w16-18TMEC mice were further

boosted with TMEC plasmids at weeks 23 and 25 (w16-18/23-

25TMEC).

Fig. 3 Successive TMEC plasmid electroporations hamper carcino-genesis even when IL-12 administration is delayed. Percentage oftumor-free mice (A) and tumor multiplicity (B) in the 7wIL12 + w16-18/23-25TMEC (w , 10 mice), 14wIL12 + w16-18/23-25TMEC (n,6 mice), and 14wIL12 + w16-18TMEC (4, 7 mice) groups. Arrow,week in which IL-12 was injected; arrowheads, week in which plasmidswere electroporated. The tumor-free survivals in 7wIL12 treated + w16-18/23-25TMEC and 14wIL12 + w16-18/23-25TMEC mice aresignificantly different (Mantel-Haenszel test, P < 0.0008), as are thoseof 14wIL12 + w16-18TMEC and 14wIL12 + w16-18/23-25TMEC mice(Mantel-Haenszel test, P < 0.008). The mean tumor multiplicity issignificantly lower in 14wIL12 + w16-18/23-25TMEC mice than in14wIL12 + w16-18TMEC mice between weeks 36 and 40 (Student’s ttest, P < 0.05). Bars, SE of the mean of tumor multiplicity. Theseexperiments were done twice.




In a neoadjuvant setting, IL-12 treatment began when mice

already displayed invasive breast cancer (14wIL12) and kept

f70% of mice tumor-free; mice were subsequently vaccinated

and boosted (w16-18/23-25TMEC plasmid electroporated mice;

Fig. 3). In addition, the combination of neoadjuvant 14wIL12with

week 16 and 18 vaccination (w16-18TMEC) kept 30% of mice

tumor-free. No inhibition of tumor takes and multiplicity was

found in mice electroporated with pcDNA3 control plasmid at

week 16 and 18 only or in combinationwith 14wIL12 (not shown).

Interleukin-12 Antiangiogenesis. Several immune fea-

tures were compared among the mice of the various treatment

groups to tease apart the immune events that made the DNA

electroporation so effective in mice treated with IL-12. These

comparisons were made within the 20th and 22nd week of age

when whole mounts display the most notable inhibition of tumor

progression in treated mice (Fig. 2C).

We have previously shown that IL-12 inhibit the angiogenic

activity of Her-2+ tumor cells in vitro (18) and in vivo (4). To

assess the weight of the antiangiogenic activity of 7wIL12, the

amount of vascular endothelial growth factor in the sera of

variously treated mice was evaluated (Fig. 4A). Whereas a slight

decrease in serum vascular endothelial growth factor was found

in mice that received 7wIL12 only, the decrease was stronger

when 7wIL12 treatment was combined with w16-18TMEC

electroporations. This antiangiogenic effect was also notably

evident in the whole mounts of mammary glands done after the

perfusion of blood vessels with India ink. In 16-week-old mice,

7wIL12 alone leads to a marked decrease of small vessel

network (20.85 F 6.48% versus 36.63 F 8.88% of total image

area F SD for 7wIL12 and control respectively; P < 0.003),

associated with a reduction in size and number of neoplastic

lesions (Fig. 4D versus B). Four weeks later, w16-18TMEC

electroporation alone lead to a shrinkage of the tumor mass,

whereas a vascular network is evident not only around the

neoplastic lesions but also in the fat pad stroma (Fig. 4C). A

strikingly impressive synergistic antiangiogenic (15.29 F 5.43

versus 41.52 F 6.14% of total image area F SD for 7wIL12

treatment + w16-18TMEC and w16-18TMEC alone respectively;

Fig. 4 Antiangiogenic effect of IL-12treatment + 16-18TMEC in 22-week-oldmice. Level of vascular endothelial growthfactor in the sera of variously treated miceas assessed by ELISA. Each determinationwas done on the sera of six mice individ-ually tested (A). B-E, Perfusion of bloodvessels with India ink followed by wholemount of the mammary glands. A wide-spread vascular network of first-ordervessels running longitudinally parallel tothe mammary ducts and numerous second-order vessels originating from first-ordervessels and penetrating the large neoplasticlesions are evident in control mice (B).7wIL12 alone markedly reduces this net-work and reduces the size and number ofneoplastic lesions (D). w16-18TMEC aloneslightly decreases lesion size and anincrease of small caliber vessels andvascular network is evident around thelesions and in the fat pad stroma (C).7wIL12 + w16-18TMEC leads to a strik-ingly impressive antiangiogenic effect.Small vessels disappear leaving a networkmostly composed of large vessels thataccompany breast ducts. Tumor lesionsare not evident (E). Three different micewere used for each treatment.




P < 0.0001) and an antitumor effect resulted from the

combination of 7wIL12 treatment + w16-18TMEC electro-

poration. Small vessels and neoplastic lesions are no more

evident, whereas the vascular mammary network is mostly

constituted by large vessels (Fig. 4E).

Spleen Germinal Centers and Antibody Response. At

20 weeks of age, the white pulp GC in the semiserial sections of

the spleen of 7wIL12 + w16-18TMEC mice were 10 F 2 per

section and 7 F 2 per section in mice that only received w16-

18TMEC. In both cases, GC were significantly more numerous

than in mice that received 7wIL12 (3 F 1 per section) or w16-

18pcDNA3 only (2 F 1 per section; P < 0,001). In addition, a

wider lymphoid white pulp with more expanded B-cell areas and

marked B-cell scattering within the T-cell zone are evident in

spleens of w16-18TMEC and 7wIL12 + w16-18TMEC mice

(Fig. 5B and C). These morphologic findings on B-cell activation

correspond to the serum anti-r-p185neu antibody titers. 7wIL12 +

w16-18TMEC mice displayed an earlier, stronger, and much

more sustained antibody response to p185neu than w16-18TMEC

mice (Fig. 5D). Following w16-18TMEC, IgG2a and IgG3 were

the most expanded isotypes and IgG1 was the least (Fig. 5E).

7wIL12 + w16-18TMEC did not induce an obvious isotype

switch, but markedly expanded the production of all isotypes. As

these isotypes mediate antibody-dependent cellular cytotoxicity,

their ability to guide Spc against r-p185neu+ TUBO cells was

assessed. Whereas no lysis is observed when TUBO cells are

incubated with sera from 7wIL12 or w16-18pcDNA3 mice, those

incubated with sera from 7wIL12 w16-18TMEC mice are lysed

Fig. 5 Effect of 7wIL12 +w16-18TMEC on the anti-r-p185neu B-cellresponse. Expression of CD45R/B220 B cell marker (red staining) inspleens from 20-week-old untreated(A), w16-18TMEC (B), and 7wIL12+ w16-18TMEC mice (C ). Thelymphoid white pulp is wider withmore expanded B-cell areas andfrequent GC in the spleen from w16-18TMEC (B) and 7wIL12 + w16-18TMEC (C) mice. D, anti-p185neu

antibody in the sera of w16-18TMECand 7wIL12 + w16-18TMEC mice.The titer is significantly higher in thelatter group than in w16-18TMECelectroporated mice, where the titerwas significantly higher (P < 0.0025at week 24 and P < 0.0035 at week35). Sera were tested at 1:100; finalresults in Ag/mL have been adjustedfor dilution. E, isotypes of anti-r-p185neu antibody in the sera (1:20)of w16-18pcDNA3 (white line), w16-18TMEC (green line), and 7wIL12 +w16-18TMEC (red line) mice. F,percentage of lysis of r-p185neu targetcells in one of three independentantibody-dependent cellular cyto-toxicity tests done with pools of serafrom five mice bled at week 20. Threedifferent mice were used for eachtreatment.




in a significantly higher percentage (P < 0.006) than those

incubated with sera from w16-18TMEC (Fig. 5F).

To further assess the significance of antibodies in the

inhibition of Her-2 carcinogenesis, BALB-neuT mice with an

impaired ability to produce them (BALB-neuT/AKOmice; ref. 18)

received 7wIL12 + w16-18TMEC (Fig. 6). The kinetics of

mammary carcinogenesis in these mice is similar to that of

untreated BALB-neuT mice. Even so, 7wIL12 + w16-18TMEC

did not elicit any detectable anti-r-p185neu antibody responses (not

shown), nor did it inhibit the onset of carcinoma (Fig. 6A),

although it led to a marginal and temporary decrease of tumor

multiplicity (Fig. 6B).

Spleen Dendritic Cells and T-Cell Activation. At 20

weeks of age, immunohistochemical examination of the semi-

serial sections of the spleen of untreated mice (Fig. 7A) or

w16-18pcDNA3 mice (not shown) displays CD11c+ cells with

dendritic cell (DC) morphology, mainly at the edge of T-cell

areas. These DCs rarely express the B7.2 (CD86) activation

marker. 7wIL12 alone gives rise to a marked DC increment and

induces preferential CD86+ DC localization at the edge of the

T-cell area (Fig. 7D), whereas the numerous DCs in the spleen

of w16-18TMEC are also scattered along the whole T-cell

areas. However, the CD86 activation marker is still preferen-

tially expressed by DC at the edge of the areas (Fig. 7E).

7wIL12 + w16-18TMEC mice synergistically increase the

number of CD86+ DC at the edge and inside the T-cell areas

(Fig. 7J and K). CD40L is a CD4 T-cell activation marker

almost absent in the spleens of untreated (Fig. 7C), 7wIL12

Fig. 6 Effects of 7wIL12 + w16-18TMEC and the transfer of immune sera on the progression of carcinogenesis in BALB-neuT mice deficient forantibody (BALB-neuT/AKO) and IFNg (BALB-neuT/IFN-gKO) production. BALB-neuT/AKO: percentage of tumor-free mice (A) and tumormultiplicity (B) of tumors in untreated (o, 8 mice) and 7wIL12 + w16-18TMEC mice (., 9 mice) and in 7wIL12 + w16-18TMEC mice that received atweek 17 and 19 serum from BALB-neuT 7wIL12 + w16-18TMEC mice (!, 5 mice). Between weeks 22 and 23, 7wIL12 + w16-18TMEC micedisplayed a tumor multiplicity that is similar to that of untreated mice but significantly higher (P < 0.04) than that of 7wIL12 + w16-18TMEC mice thatadditionally received immune serum. BALB-neuT/IFN-gKO: percentage of tumor-free mice (C) and tumor multiplicity (D) in untreated (o, 12 mice),7wIL12 + w16-18TMEC mice (., 5 mice), and in 7wIL12 + w16-18TMEC mice that received at week 17 and 19 serum from BALB-neuT 7wIL12 +w16-18TMEC mice (!, 5 mice). 7wIL12 + w16-18TMEC mice displayed a tumor multiplicity that is significantly lower of that of untreated mice (P <0.0001-0.02) between weeks 19 and 39 but higher of that of 7wIL12 + w16-18TMEC mice that also received immune serum (P < 0.03) between weeks23 and 25. Bars, SE. These experiments were done twice.




(Fig. 7F ), and w16-18pcDNA3 mice (not shown), but

frequently expressed in those of w16-18TMEC mice (Fig. 7I)

and even more in 7wIL12 + w16-18TMEC mice, where it is

mostly colocalized with CD86+ DC (Fig. 7L).

When the production of IFN-g by Spc against p185neu+

TUBO cells was evaluated with the cytokine secretion assay

(Miltenyi Biotec), the frequency of both CD4+ and CD8+ cells

producing IFN-g was much higher in Spc from 7wIL12 + w16-

18TMEC (Table 1). This pattern of IFN-g release correlates with

their higher cytotoxic activity compared with all the other

treatment groups.

To further assess the weight of IFN-g, BALB-neuT/

IFNgKO mice (13) received 7wIL12 + w16-18TMEC (Fig. 6).

The kinetics of mammary carcinogenesis in these mice is similar

Fig. 7 Effect of 7wIL12 + w16-18TMEC on DC and T-cell activation markers. Expression of CD11c, CD86, and CD40L (red staining) in the spleenof 20-week-old untreated and treated BALB-neuT mice. In untreated mice, CD11c-positive cells (DC) are present in the T-cell area and particularly at itsedge (A) where expression of the activation marker B7.2 (CD86; B) is rare. Activated T cells (CD40L+) are almost absent (C). 7wIL12 treatmentprovokes a marked increment of DC, which are preferentially localized at the border of the T-cell area (D) and frequently CD86+ (E). CD40L+ arescarce (F). In w16-18TMEC mice, DCs are more represented than in untreated mice and are found at the edge and scattered throughout the T-cell area(G). The activation marker CD86 is preferentially expressed by the borderline DC (H). CD40L+ are frequently present and scattered within the T area(I). The combined treatment has a synergistic effect on the number and distribution of DC. They are impressively represented (J) and diffuselyactivated (K), even in the inner zone of the T-cell area. CD40L+ cells are numerous and scattered within the T area (L). Magnification: A , B , D , E ,G , H , J , K , �400; C , F, I , L �630). Three different mice were used for each treatment.

Table 1 Effect of 7wIL12 + w16-18TMEC on the IFN-g production and cytotoxic activityagainst p185neu+ TUBO cells

w16-18pcDNA3electroporation

w16-18TMECelectroporation

7wIL12treatment

7wIL12 treatment +w16-18TMECelectroporation

Percentage of lymphocytesTotal CD4+* 34 F 6 21 F 2 27 F 1 23 F 4Total CD8+ 10 F 3 7 F 1 8 F 1 7 F 1CD4+ releasing IFN-g+ 23 F 7 37 F 7 16 F 15 73 F 15CD8+ releasing IFN-g+ 29 F 7 27 F 3 30 F 4 71 F 16Cytotoxicity (LU20y) against p185neu+TUBO cells

0.8 F 0.5 0.5 F 0.2 10.9 F 3 41.1 F 7

*Mean F SE of the percentage of positive cells as assessed by three independent experiments of flow cytometry.yMean F SE of LU20 of Spc from three mice from each treatment group following specific in vitro stimulation (7wIL12 versus 7wIL12 + w16-

18TMEC, Student’s t test, P < 0.05).




to that of untreated BALB-neuT mice. This combination did not

delay the onset of carcinomas (Fig. 6C), but tumor multiplicity

was transiently reduced (Fig. 6D).

Adoptive Transfer. Whole mounts of the mammary

glands illustrate the stage of carcinogenesis in BALB-neuT

mice. This method was exploited as an intermediate end point to

assess the protection provided by the adoptive transfer of

antibodies and T lymphocytes. Serum or Spc T cells (90-92%

CD90+), isolated by magnetic cell sorting with the autoMACS

magnetic separator (Miltenyi Biotec), were collected from

groups of 20-week-old untreated control and from 7wIL12 +

w16-18TMEC mice. Recipient, otherwise untreated BALB-neuT

mice received serum i.p. and T cells i.v. on week 10 and 12 and

the whole mounts of their mammary glands were prepared at

week 16. The adoptive transfer of T cells and (to a greater extent)

serum significantly inhibited carcinogenesis progression

(Table 2). The inhibition achieved by the combined transfer of

sera and T cells was not significantly different from that obtained

with serum alone.

The substantial role of antibodies was also evident in

BALB-neuT/AKO and BALB-neuT/IFNgKO mice that received

the adoptive transfer of immune serum. In both lines of KO mice,

immune serum significantly reduced tumor multiplicity (Fig. 6).

Modulation of T Regulatory Cells. As CD4+/CD25+

T regulatory cells with suppressive activities are used by the

immune system to control its response to self-antigens (23), we

investigated their fluctuation during IL-12 treatment and

specific immunization. Twenty-week-old w16-18pcDNA3 mice

and w16-18TMEC have 14.06 F 0.49% and 14.16 F 0.33%

CD4+/CD25+ cells. This number was slightly, but significantly,

decreased in 7wIL12 mice (12.65 F 0.132) and not further

decreased (12.597 F 0.129) by 7wIL12 + w16-18TMEC

(Fig. 8).

DISCUSSION

In a preclinical model of multifocal mammary carcinomas,

the combination of IL-12 as a neoadjuvant therapeutic agent

followed by ‘‘immune-surgery’’ of the residual tumor cells

through DNA vaccine electroporation seems able to produce a

sustained cure. DNA vaccine electroporation during the early

stages of carcinogenesis protects BALB-neuT mice predestined

to die of mammary carcinomas (18), whereas later vaccination is

totally ineffective. However, combination of early systemic

administration of IL-12 with late electroporation provides

sustained control of invasive carcinomas, a result that has never

been even approached in this devastating model of autochtho-

nous cancer affecting all 10 mammary glands.

The curative potential of this combination seems to be the

outcome of the ability of IL-12 to both hamper early carcinogen-

esis and enhance the specific antitumor immune response (5, 10,

11, 24). The enhancement by IL-12 of the specific protective

response elicited by DNAvaccine electroporation fits in well with

its powerful adjuvant activity observed with a variety of vaccines

against infectious diseases (25). Indeed, induction of IL-12 release

is an important mechanism whereby adjuvants exert their effects

(26, 27). IL-12 activates innate immune cells and promotes their

production of cytokines and chemokines, and thus mediates local

recruitment of cells of innate and adaptive immunity (28).

Furthermore, an IL-12-conditioned microenvironment is suitable

for APC activation, antigen presentation, and prevention (or

reversal) of the induction of tolerance toward tumor antigens (29).

In addition, the delay of progression observed when

mammary glands display widespread atypical hyperplasia is in

line with IL-12-dependent impairment of tumor-driven angio-

genesis. The marked inhibition of vascular endothelial growth

factor production by 7wIL-12 treatment was somewhat expected,

as we have previously observed that IL-12-activated lymphoid

cells change gene and protein expression of Her-2 tumor cells

and down-modulate their angiogenic activity (8, 10, 11, 30). This

inhibition correlates with the dramatic inhibition of the network

of small vessels associated with mammary lesions and their

hampered progression in 7wIL12 mice. Early IL-12 treatment

seems to slow the progression of the incipient tumor and

consequently allows the immune response to be more effective

against a slow-growing tumor. The significance of tumor

angiogenesis as a prognostic indicator has been documented in

various kinds of human tumors (31, 32). Tumor vascularity is an

indicator of aggressiveness in breast cancer that significantly

correlates with its clinical and histologic grades (33).

We have previously shown that an antibody response (5, 8)

and production of IFN-g (13, 15) are essential for successful

preventive vaccination. Our present data indicate that combina-

tion of IL-12 with DNA electroporation does not obviate the

need for an active IFN-g and antibody response, but markedly

enhances the number of T cells that specifically release IFN-g in

the presence of Her-2+ target cells, the number of spleen GC, the

titer of anti-Her-2 antibodies, and the antibody-dependent

cellular cytotoxicity. Parallel experiments on wild-type BALB/

c mice also showed that 7wIL12 + w16-18TMEC mice produced

an anti-Her-2 antibody titer 2-fold of that of mice that received

w16-18TMEC only (data not shown). Whereas many of the

Table 2 Mammary carcinogenesis in recipient BALB-neuT mice to which T cells and serum from7wIL12 + w16-18TMEC mice were adoptively transferred

Mean lesion size(Am) F SD

Number of lesions>300 Am F SD Tumor index

Ctrl 246 F 32 14 F 12.8 3,444T cells 169 F 37 3.1 F 1.7 524Serum 102 F 43 1.16 F 0.75 118T cells + serum 127 F 51 1.33 F 1.2 169

NOTE. Values of experimental groups are significantly different from control mice (P V 0.0002) and T-cell treatments are significantly differentfrom serum treatment (P V 0.003). At least 30 mammary glands of 16-week-old recipient BALB-neuT mice were evaluated in each treatment group. Themean lesion sizes were measured by computer-assisted image analysis in at least 100 randomly selected points without lesions. The tumor index is theproduct of the mean lesion size and the mean lesion number.




downstream factors triggered by systemic IL-12 activate several

immune reaction mechanisms and hamper tumor growth (28),

IL-12-dependent activation of DC may make a critical

contribution to an effective anti-Her-2 response (7). The DC

influx and activation at the edge of T-cell areas parallel an

increase in activated CD4+ CD40L+ T cells, which release IFN-g

(and probably other cytokines), leading to GC formation and

expansions. Moreover, many features of BALB-neuT mice

indicate that they are tolerant to the rp185neu (18). Because

CD4+/CD25+ T cells with suppressive activities constitute one of

the mechanisms through which the immune system maintains

tolerance, we determined whether vaccination schedule reduce

their number. Mice treated with IL-12, both alone or in

combination with TMEC, display a slight but significant

decrease of CD4+/CD25+ Spc that may contribute to the

generation of a good anti-p185neu immune response.

Previous work with transplantable tumors has shown that

inhibition of tumor angiogenesis by IL-12 rests on its ability to

elicit secondary cytokines and tertiary chemokines and mono-

kines that activate the endothelial cells of neoformed tumor

vessels and facilitate leukocyte extravasation (10). These very

downstream factors activate innate immunity, enhance the

priming of adaptive responses, activate leukocyte subsets that

produce proinflammatory cytokines, and induce polymorphonu-

clear cells to destroy newly formed tumor vessels (28, 30).

Combination of distinct antiangiogenic mechanisms intrinsically

intermingled with IL-12 immunomodulatory activities leads to

the inhibition of both Her-2-dependent tumors in transgenic mice

and chemically induced carcinogenesis (24). The resulting delay

in tumor progression enables late DNA electroporation to be as

effective as early vaccination, as the burden of late mammary

lesions is confined to its initial stage. More than 60% of the

transgenic mice kept tumor-free by IL-12 in combination with

TMEC plasmid electroporation were able to reject a lethal

challenge of p185neu+ syngenic TUBO cells done 15 weeks after

the last DNA vaccination, indicating that this combined

immunization also induces a significant and sustained memory

response (data not shown).

Early stimulation of innate immunity in humans equivalent

to that elicited by IL-12 in mice could delay the progression of a

diffuse preneoplastic lesion and allow identification of the target

antigens it expresses and the manufacturing of a specific vaccine.

Progression will be impeded and the protective potential of the

vaccine itself could be significantly enhanced.

ACKNOWLEDGMENTSWe thank Prof. John Iliffe for editing the manuscript.

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2005;11:1941-1952. Clin Cancer Res Michela Spadaro, Elena Ambrosino, Manuela Iezzi, et al. ImmunityInterleukin-12) and Adaptive (DNA Vaccine Electroporation)through Sequential Stimulation of Innate (Neoadjuvant Cure of Mammary Carcinomas in Her-2 Transgenic Mice

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